1. Field of the Invention
The present invention relates to methods for detecting one or more protein conformers in a sample containing a protein having at least two protein conformations. Thus, the invention is directed to methods for distinguishing and quantitating stable protein conformations of the same protein. An example of a protein which is known to exist in at least two conformations is the normal prion protein (PrPC) and its infectious isoform (PrPSc).
2. Description of the Art
The vast majority of proteins adopt only one stable conformation, however, exceptions exist. Indeed, in recent years it has come to light that many proteins can adopt more than one stable folded conformation that is they have multiple isoforms or conformers. Interestingly, a number of the proteins having multiple isoforms are important agents of disease see e.g., Citi, F., and Dobson, C. M. (2006) Ann. Rev. Biochem. 75:333-366 and Wickner R B., et al. (2008) Bioessays. 2008; 30:955-64.
At least sixteen types of human disease are associated with fibrils made of abnormally folded proteins (see e.g., Pepys (1996) Amyloidosis. In Weatherhall, D. J, Ledingham, J. G. G. and Warell, D. A. (Eds), The Oxford Textbook of Medicine, 3rd edition, Vol 2, Oxford University Press, Oxford, UK, 1512-1524). For example, amyloid fibrils are associated with diseases including, but not limited to spongiform encephalopathies, Alzheimer's disease, Parkinson's disease, Huntington's disease, and type II diabetes.
An example of a protein which causes disease when misfolded is the prion protein. Prions, such as mammalian PrP and fungal sup35, are unique amongst amyloidogenic proteins in that they are known to exist in more than two stable conformations. Prion diseases have properties that are maintained upon transmission from one host to the next, allowing different prion “strains” to be distinguished. The strains cause specific phenotypes, such as different symptomology (ataxias, hyperactivity, lethargy), time from exposure to disease, and different tissue distribution of PrPSc.
A critical difference between prions and other amyloids is that prions are by definition infectious (see e.g., Prusiner (1982) Science 216(9):136-144). Very substantial and diverse evidence suggests that transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases affecting humans and animals, are mediated by a prion, named PrP (prion protein) (see e.g., Prusiner (1982) supra; Prusiner (1998) Proc. Natl. Acad. Sci. 95:13363-13383; Soto and Castilla (2004) Nat. Med. 7 Suppl. S63-S67; Aguzzi and Polymenidou (2004) Cell 116:313-327; and Prusiner (1991) Science 252:1515-1522). The most widely studied TSEs in food-producing animals include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in mule deer and elk. Other TSEs in animals included transmissible mink encephalopathy (TME) in mink and feline spongiform encephalopathy (FSE) of cats. Prion diseases of humans have also been identified. These include: Creutzfeldt-Jakob Disease (CJD); Gerstmann-Straussler-Scheinker Syndrome (GSS); Fatal Familial Insomnia (FFI), and Kuru.
PrP exists in at least two conformations, PrPC and PrPSc. The latter is associated with TSEs, and PrP showing the physico-chemical characteristics of PrPSc is isolated as the main and most probably only component of the TSE infectious agent. PrPC can be converted into PrPSc, in the presence of pre-formed PrPSc, through a poorly understood molecular process (see e.g., Aguzzi and Polymenidou (2004) supra; Prusiner (1991) supra; and Come et al. (1993) Proc. Natl. Acad. Sci. 90:5959-5936). The structure of PrPC has been characterized by NMR (see e.g., Riek et al. (1996) Nature 382:180-182), but that of PrPSc is largely unknown, as its insolubility in non-denaturing solvents has seriously hampered analytical efforts. It is known, however, that PrPSc and PrPC differ with respect to secondary, tertiary and quaternary structures (see e.g., Prusiner (1998) supra). No covalent differences have been detected between the two molecules (see e.g., Stahl et al. (1993) Biochemistry 32:1991-2002), although the possibility that post-translational modifications of a small set of PrPC molecules could trigger structural changes relevant to initiation of conversion to PrPSc cannot be ruled out (see e.g., Requena et al. (2001)). Studies using Fourier transform infrared spectroscopy (FTIR) indicate that PrPSc contains an increased fraction of β-sheet and decreased fractions of α-helix and random coil with respect to PrPC (see e.g., Prusiner (1998) supra). PrPC is a monomeric protein anchored to the cell membrane through a glycan phosphoinositol (GPI) anchor. In contrast, PrPSc is isolated as an aggregate. PrPSc is partially resistant to proteinase K (PK), that trims an amino terminal segment of the protein generating a well defined resistant core termed PrP 27-30 whereas PrPC is rapidly degraded by PK (see e.g., Prusiner (1998) supra). PrP 27-30 retains the infectious character, and hence the essential structural characteristics, of PrPSc, with the trimmed aminoterminal domain probably consisting of a highly flexible tail as seen in PrPC. In the presence of detergent, PrP 27-30 further polymerizes to rod-shaped filaments with the tinctorial properties of amyloid (see e.g., McKinley et al. (1991) J. Virology 65(3): 1340-1351).
At present, protein conformers can be discriminated by methods such as (FTIR) or circular dichroism (CD) spectroscopy, but only when the proteins have been extensively purified. NMR and X-ray diffraction, the most common methods to determine protein structure, have been used successfully to determine the three dimensional structure of the soluble forms of amyloidogenic proteins (e.g., the cellular form of the prion protein PrPC), however these methods can not be used for the amyloids themselves since amyloids by nature are neither soluble, as required by NMR, nor crystallizable, as required by high resolution X-ray diffraction.
Considerable effort has gone towards attempts to find antibodies that discriminate PrPSc from PrPC but to date, no antibodies have been found that selectively bind PrPSc see e.g., Gibbs C J, Gajdusek D C, Morris J A. Viral characteristics of the scrapie agent in mice. In: Gajdusek C J, Gibbs C J, Alpers M P, eds. Slow, Latent, and Temperate Virus Infections. Washington, D. C.: U.S. Government Printing Office; 1965:195-202; Porter D D, Porter H G, Cox N A. (1973) J Immunol. 111:1407-1410; Prusiner, S. B. Prions: (1984) Adv. Virus. Res. 29:1-56; Gardash'yan, A M, Nartsissov N V, and Bobkova O V. (1971) Bull. Exp. Biol. Med. 1971; 71: 664-666; Moulton J E, Palmer A C. (1959) Cornell Vet. 49:349-359; Gardiner A C. (1966) Res Vet Sci. 1966; 7: 190-195; Chandler R L. Vet Rec. (1959) 71:58-59; Pattison I H, Millson G C, Smith K. (1964) Vet. Sci. 1964; 5: 116-121; Clarke M C, Haig D A. (1966) Vet. Rec. 78:647-649; Gibbs C J Jr. (1967) Curr. Top. Microbiol. Immunol. 40:44-58.
In view of the considerable human and animal health considerations related to alternately folded proteins that form amyloid deposits, what is needed are methods to detect, distinguish, and, if desired, quantitate two or more protein conformations of the same protein.